A standardized procedure to obtain mesenchymal stem/stromal cells from minimally manipulated dental pulp and Wharton's jelly samples.

Transplantation of mesenchymal stem/stromalcells (MSCs) has emerged as an effectivemethod to treat diseased or damagedorgans and tissues, and hundreds of clinicaltrials using MSCs are currently under way todemonstrate the validity of such a therapeuticapproach. However, most MSCs used for clinicaltrials are prepared in research laboratorieswith insufficient manufacturing quality control.In particular, laboratories lack standardizedprocedures for in vitro isolation of MSCs fromtissue samples, resulting in heterogeneouspopulations of cells and variable experimentaland clinical results.MSCs are now referred to as Human CellularTissue-based Products or Advanced TherapyMedicinal Products, and guidelines fromthe American Code of Federal Regulation ofthe Food and Drug Administration (21 CFRPart 1271) and from the European MedicinesAgency (European Directive 1394/2007) definerequirements for appropriate production ofthese cells. These guidelines, commonly called"Good Manufacturing Practices" (GMP),include recommendations about laboratorycell culture procedures to ensure optimal reproducibility,efficacy and safety of the finalmedicinal product. In particular, the Food andDrug Administration divides ex vivo culturedcells into "minimally" and "more than minimally"manipulated samples, in function of theuse or not of procedures "that might alter thebiological features of the cells". Today, minimalmanipulation conditions have not beendefined for the collection and isolation ofMSCs (Torre et al. 2015)(Ducret et al. 2015).Most if not all culture protocols that have beenreported so far are unsatisfactory, becauseof the use of xeno- or allogeneic cell culturemedia, enzymatic treatment and long-termcell amplification that are known to alter thequality of MSCs.The aim of this study was to describe a standardizedprocedure for recovering MSCs withminimal handling from two promising sources,the dental pulp (DP) and the Wharton's jelly(WJ) of the umbilical cord. The quality and homogeneityof the expanded cell populationswere assessed by using flow cytometry withcriteria that go beyond the International Societyof Cellular Therapy (ISCT) guidelines forMSC characterization.

O34-pathogen sensing by human odontoblasts.

Human odontoblasts are neural crest-derived, dentin-producing mesenchymal cells aligned at the periphery of the dental pulp. They become exposed to cariogenic oral bacteria as these progressively demineralise enamel then dentin to gain access to the pulp. Due to their situation at the dentin-pulp interface, odontoblasts are the first cells encountered by invading pathogens and/or their released components, and represent, in the tooth, the first line of defence for the host. Previous studies have shown that odontoblasts are able to sense pathogens and elicit innate immunity. In particular, they express several pathogen recognition receptors of the Toll-like receptor (TLR) and nucleotide-binding oligomerisation domain (NOD) families, which allow them to recognize specific bacterial and viral components. So far, most studies aiming at elucidating the role of odontoblasts in the dental pulp innate response have focused on Gram-positive bacteria, as these largely dominate the carious microflora in initial and moderate dentin caries lesions. In vitro, odontoblasts were found to be sensitive to Gram-positive bacteria-derived components, mainly lipoteichoic acid which is recognized through cell membrane TLR2. Our studies have shown that engagement of odontoblast TLR2 by LTA triggers TLR2 and NOD2 up-regulation, NF-B nuclear translocation, production of various chemokines including CCL2, CXCL1, CXCL2, CXCL8 and CXCL10, while promoting immature dendritic cell recruitment. Conversely, LTA down-regulates major dentin matrix components, including collagen type I and dentin sialophosphoprotein, as well as TGF-b1, a known inducer of dentin formation. We provide here additional data showing the fine localization of NOD2 in healthy dental pulps, as well as differential regulation of TLR2, TLR4, NOD2, CCL2 and CXCL8 genes by LTA and the synthetic TLR2 agonists Pam2CSK4 and Pam3CSK4. It appears from the aforementioned data that odontoblast-triggered immune events constitute potential targets for interrupting the signaling cascades which lead to excessive immune response and necrosis in the dental pulp tissue challenged with cariogenic bacteria. In particular, preventing Gram-positive bacteria recognition or signal transduction by pattern recognition receptors may represent a valuable strategy to achieve this goal. Future studies in the field will pave the way for designing novel therapeutic agents which modulate odontoblast behaviour to promote pulp healing and repair.

Effects of fluctuating moisture and temperature regimes on the persistence of quiescent conidia of Isaria fumosorosea.

Conidia of Isaria fumosorosea were submitted to three regimes of temperature and moisture to simulate microclimatic conditions which prevail in temperate (43% RH and 28 degrees C to 98% RH and 15 degrees C), subtropical (75% RH and 35 degrees C to 98% RH and 25 degrees C), and arid areas (13% RH and 40 degrees C to 33% RH and 15 degrees C) with daily fluctuating cycles. Germination, conidial viability, and virulence to Spodoptera frugiperda larvae were less affected after 20 days exposure under temperate cycling conditions than under arid and subtropical conditions. Exposure of conidia for 20 days to constant nocturnal simulated conditions of any tested regime weakly affected conidial persistence, whereas diurnal conditions exerted the most detrimental effects of high temperatures. However, when tested at both 45 degrees C and 50 degrees C at 33% RH for 160 h, the persistence of I. fumosorosea conidia was relatively higher than expected. These results emphasize that climatic conditions prevailing in environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.

Influence of temperature preferences of two Paecilomyces fumosoroseus lineages on their co-infection pattern.

In order to clarify the epidemiological potential of entomopathogenic fungi for insect pest control, the role of the temperature as one environmental constraint was investigated on the pattern of co-infection of Galleria mellonella by two distinct lineages of a hyphomycete, Paecilomyces fumosoroseus. The distribution of conidial populations collected on cadavers of hosts co-infected under 20 regimes, ranging from 13 to 35 degrees C, was examined. The apparent temperature tolerance of both fungal isolates was related to their in vitro colony growth and their in vivo sporulation ability. The conidial populations were characterized by molecular markers based on restriction fragment length polymorphisms of the internal transcribed spacers (ITS-RFLP) and random amplified polymorphic DNA (RAPD) contrasting profiles in combination with the conidial size. This study allowed a different temperature profile was identified for each isolate. Under most temperature regimes, only one lineage prevailed upon the infected insect; whereas both lineages coexisted at 20-25 and 25-25 degrees C. When one haplotype dominated, the displacement of the other one depended on its temperature tolerance. These results suggest that more consideration should be given to population-genetics analyses for evaluating the adaptability of microbial control agents to targeted environments.

Effects of fluctuating moisture and temperature regimes on the infection potential of Beauveria bassiana for Rhodnius prolixus.

The effect of both moisture and temperature on the infective potential of Beauveria bassiana to the Chagas' disease vector, Rhodnius prolixus, was studied under fluctuating regimes. At constant 25 degrees C, contaminated first-instar nymphs exposed to increasing daily periods of initial exposure to 97% RH, followed by transfer to reduced humidity (43, 53, 75, and 86% RH), showed a significant reduction in mortality when the 97% RH exposure time declined from 12 to 8 h per day. The duration of disease incubation depended on the daily 97% RH exposure time. Under fluctuating regimes of both humidity (97% RH versus 75% RH) and temperature (15/28, 20/25, 25/28, and 25/35 degrees C), first-instar mortality was affected by weather conditions, daily 97% RH exposure time (8, 12, and 16 h per day), and number of temperature and humidity fluctuations before transferring tested insects to constant unfavorable conditions. In most cases, at 12/12 h alternating cycles, high and rapid mortality required five cycles. Under these fluctuating regimes, fungus-induced mortality and mortality time were similarly affected in third- and fifth-instar nymphs by the daily 97% RH exposure time. Despite a lower susceptibility of older larval stages, mortality rates in insects exposed for at least 12 h per day at 97% RH remained very high except at 15 degrees C. Moisture and temperature regimes at 12/12 h cycling significantly affected the dose-mortality response in first-instar nymphs. The most favorable conditions consisted of 97%-20 degrees C combined with either 75%-25 degrees C or 43%-25 degrees C. Under less favorable alternating conditions (lower and higher temperatures) the amounts of inoculum required for killing 50% of first-instar nymphs were 10 or 20 times higher. From a vector control standpoint, daily high humidity appears to be the most crucial climatic constraint. B. bassiana has the potential to control R. prolixus populations with applications made during the rainy seasons when humidity is high.

Development of Rhodnius prolixus (Hemiptera: Reduviidae) under constant and cyclic conditions of temperature and humidity.

Development of Rhodnius prolixus after eclosion until the adult stage was studied at constant temperatures (T), 15, 20, 25, 28, 35 C, and relative humidities (RH), 75, 86 and 97%, and fluctuating (16/8 hr) temperatures, T I/II, 15/28 C, 20/25 C, 25/28 C and 25/35 C, and relative humidities, RH I/II, 86/75% and 97/75%. Eclosion or molting were not observed at 15 C and 86 or 97% RH, respectively. At 35 C and 75% RH only few insects molted. By alternating T I/II, 15/28 C and 25/35 C, insects developed at high frequency. Cumulating the average lengths of the interphases within independent groups for each instar, R. prolixus reached the adult stage most rapidly (86.7 days) and at highest frequency per instar (mean: 91.8%) at 28 C and 75% RH. Under fluctuating T I/II, development was completed within 100 days or less at 25/28 C and 25/35 C with high rates of hatch and molting. Development was slowest at fluctuating TI/II, 15/28 C and 20/25 C (>185 days), and at constant 20 C (>300 days). Mortality was higher at constant 97% RH or fluctuating RH I, 97%, than at constant or fluctuating 86% RH. Refeeding was minimal at optimal conditions of T and RH for development. The most refeeding was observed at a constant 35 C.

Dependence of the entomopathogenic fungus, Beauveria bassiana, on high humidity for infection of Rhodnius prolixus.

The impact of relative humidity (RH) on the infective potential of the isolate Bb INRA 297 of Beauveria bassiana (Bals.) Vuillemin (Deuteromycotina: Hyphomycetes) against first instar nymphs of Rhodnius prolixus Stål. (Hemiptera: Reduviidae) was determined. Fungus-treated insects were exposed to RHs ranged from 75 to 100% at 25 degrees C. Results clearly showed a threshold of humidity at ca. 96% for high and rapid mortality. After initial exposure to increasing periods of 97% (4, 8, 16, 24, 36 and 48 h) and subsequent transfer to constant lower RHs (43, 53, 75 and 86%) at a constant 25 degrees C, an incubation of at least 48 h at 97% RH was necessary to kill all insects. On changing RHs of 97/75% and different regimes of temperature (15/28 degrees C, 20/25 degrees C, 25/28 degrees C, and 25/35 degrees C), at least 72 h of initial exposure at 97% RH for the 15/28 degrees C regime, 48 h for the 20 degrees/25 degrees C and 25/28 degrees C regimes and 36 h for 25/35 degrees C were needed to kill all insects over a 6-day incubation time. Delayed exposure to favorable moisture condition (97% RH), significantly affected infection for up to a 3-day delay within the various temperature-humidity regimes tested.

Factors affecting conidial production of Beauveria bassiana from fungus-killed cadavers of Rhodnius prolixus.

The epizootic potential of Beauveria bassiana for control of the triatomine vectors of Chagas' disease was investigated by studying the effects of both biotic and abiotic factors on the recycling of a highly virulent fungal isolate from fungus-killed cadavers of Rhodnius prolixus. The conidial production of B. bassiana from mummified cadavers of R. prolixus required high RH levels of at least 96.5% RH. At 97% RH and 25 degrees C, the amount of conidia per insect ranged from 5.3 x 10(6) (on first-instar larval cadavers) to 1.7 x 10(8) (on adult cadavers) depending on the size of the different stages of development of the host. Under optimal conditions, B. bassiana sporulation from R. prolixus cadavers took place in 4 to 5 days after death. At a high humidity level (97% RH) the intensity of the conidial production from Rhodnius cadavers was little affected by temperature over a range from 15 to 25 degrees C, but it declined at 28-30 degrees C and was null at 35 degrees C. There was only a weak influence of the blood meal of nymphs and its timing on the conidial production from B. bassiana-killed cadavers. The recycling capacity of different B. bassiana isolates, selected for their pathogenic activity to R. prolixus, did not differ. From a vector control standpoint, high humidity appears to be the most crucial climatic constraint. The recycling ability of B. bassiana on fungus-killed R. prolixus might contribute to the regulation of triatomine vectors only after applications during the rainy seasons.

Inactivation of conidia of Paecilomyces fumosoroseus by near-ultraviolet (UVB and UVA) and visible radiation.

The detrimental effects of solar radiation, especially the ultraviolet waveband, on quiescent conidia of Paecilomyces fumosoroseus were investigated. Conidia were irradiated by a high-intensity source, which emitted a continuous spectrum from 270 to 1100 nm and which was equipped with long-pass filters to block short wavelengths below 280, 295, 320, or 400 nm. After irradiation, conidia were tested for germinability, survival, and infectivity toward Spodoptera frugiperda larvae. It was demonstrated that the detrimental effects of light depended on irradiance in the shortest wavelengths. The UVB (280-320 and 295-320 nm) appeared to be the most detrimental part of natural radiation, although UVA (320-400 nm) was also harmful. Visible and near infrared radiations were less harmful than UV. Our results demonstrate that the irradiance of the UVB waveband should be considered as the pertinent factor for the detrimental effects of sunlight on the persistence of conidia of entomopathogenic fungi in insolated environments.

Temperature and moisture requirements for conidial germination of an isolate of Beauveria bassiana, pathogenic to Rhodnius prolixus.

The effects of temperature, relative humidity and water activity on germination of conidia of an isolate of Beauveria bassiana (Bals.) Vuill. pathogenic to the triatomine vector of Chagas' disease, Rhodnius prolixus Stål., were investigated in vitro. Germination occurred at temperatures between 15 degrees C and 35 degrees C under saturated atmosphere and the optima ranged from 25 degrees C to 30 degrees C. At the extreme temperatures tested (15 degrees C and 35 degrees C) the germination process was delayed, but germination rates reached more than 95%. Germination of B. bassiana conidia was strongly affected by moisture conditions. The availability of water, in both atmospheric and liquid conditions, caused changes in germination times as well as in germination rates. For example, at 25 degrees C + 0.5 degrees C, germination took place within 20 h at 95.5% RH, whereas it needed 72 h of incubation at 90% RH. Germination times increased as the water activity declined from 0.96 a(w) to 0.92 a(w). Below 0.92 a(w), no germination was observed after a 72 h incubation time.

Effect of temperature on vegetative growth among isolates of Metarhizium anisopliae and M. flavoviride.

Effects of temperature on vegetative growth on a semi-synthetic medium of 22 isolates of Metarhizium anisopliae and 14 isolates of M. flavoviride were determined. The majority of isolates of both species grew between 11 and 32 degrees C; several isolates grew at 8 and 37 degrees C. None of the isolates grew at 40 degrees C. Relative growth rate, calculated from the maximum growth rate for each isolate, was significantly affected by temperature and isolate, with significant isolate * temperature interactions. The maximum absolute growth rates among the isolates ranged from 2.5 mm to 5.9 mm/day. Optimal temperatures were generally between 25 and 32 degrees C with several isolates exhibiting optimal growth at temperatures as high as 32 degrees C. Overall, relative growth rates were greater in isolates of M. anisopliae than M. flavoviride at temperatures of 25 degrees C or lower; conversely mean relative growth rates were greater in M. flavoviride than M. anisopliae at temperatures higher than 25 degrees C. However, the two most cold tolerant isolates at 8 degrees C were M. flavoviride and the three most heat tolerant at 35 degrees C were M. anisopliae. Since temperature growth responses varied considerably between isolates, strain selection according to thermal tolerance may be warranted when choosing a strain for development as a microbial control agent.

Effects of temperature and solar radiation interactions on the survival of quiescent conidia of the entomopathogenic hyphomycetePaecilomyces fumosoroseus (Wize) Brown and Smith.

The detrimental effect of solar radiation on the survival of conidia of the entomopathogenic fungusPaecilomyces fumoroseus was studied by monitoring germinability and ability to form colonies (CFU) of conidia irradiated at two temperatures, 25 and 35 °C, harmless to shaded conidia. There was no apparent effect when spores were exposed to a high level of artificial radiation (0.66 W m(-2) UVB). However, at a lower level of irradiance (0.33 W m(-2)), effects of radiation occurred more quickly at 35 °C than at 25 °C. Under natural solar radiation, the rate of decrease in germinability or viability was doubled at 35 °C as compared to 25 °C, indicating an interaction between temperature and radiation effects under natural conditions. This interaction was not detected in indoor experiments, indicating that the spectral distribution of UV radiation has to be taken in account as well as its irradiance when studying its effects.

Variability in susceptibility to simulated sunlight of conidia among isolates of entomopathogenic Hyphomycetes.

The influence of simulated sunlight on survival of conidia of 4 species of entomopathogenic Hyphomycetes was investigated. Conidia from 65 isolates ofBeauveria bassiana, 23 ofMetarhizium anisopliae, 14 ofMetarhizium flavoviride and 33 isolates ofPaecilomyces fumosoroseus were irradiated by artificial sunlight (295 to 1,100 nm at an ultraviolet-B irradiance of 0.3 W m(-2)) for 0, 1, 2, 4 and 8 h. Survival was estimated by comparing the number of colony forming units (CFU) produced by conidia exposed to irradiation to the number of CFUs produced by an unexposed control. Survival decreased with increased exposure to simulated sunlight; exposure for 2 h or more was detrimental to all isolates tested. Overall, isolates ofM. flavoviride were the most resistant to irradiation followed byB. bassiana andM. anisopliae. Conidia ofP. fumosoroseus were most susceptible. In addition to the large interspecies differences in susceptibility to irradiation, there was also an intraspecies variation indicating that strain selection to irradiation tolerance may be important in the development of microbial control agents where increased persistence in an insolated environment is desirable.

Relative susceptibility of different stages of Rhodnius prolixus to the entomopathogenic hyphomycete Beauveria bassiana.

Laboratory bioassays were conducted to determine the relative susceptibility of eggs, 1st-, 3rd-, 5th-instar nymphs and adults of Rhodnius prolixus to one isolate of the entomopathogenic hyphomycete, Beauveria bassiana. Treatments consisted of directly spraying on insects of increasing doses of inoculum (3 x 10(2) to 3 x 10(5) conidia per cm2). Mortality due to all doses of conidia was very high in the five tested stages of the target insect. Experiments on eggs demonstrated that the fungal isolate was able to kill eggs before they hatched. Both time-mortality and dose-mortality responses showed that the susceptibility of R. prolixus varied according to its stage of development and increased with age. As a matter of fact, at the dose of 3 x 10(3) conidia per cm2, LD 50 varied between 11.2 days in 1st-instar nymphs and 6.4 days in both 5th-instar nymphs and adults. Comparison of LD50 permitted to estimate that 1st-instar nymphs were about 700-fold less susceptible than the two oldest stages.

[Method of biological control of Triatominae, vectors of Chagas disease, using entomopathogenic Hyphomycetes. Preliminary study]. / Mise au point d'une méthode biologique de lutte contre les Triatominae, vecteurs de la maladie de Chagas avec des hyphomycètes entomopathogènes. Etude préliminaire.

Bioassays determined the pathogenic activity of 14 strains of 5 entomopathogenic hyphomycetous species (Fungi imperfecti), Beauveria bassiana, Beauveria brongniartii, Metarhizium anisopliae, Nomuraea rileyi and Paecilomyces fumosoroseus to Rhodnius prolixus. Treatments consisted of direct spraying with conidial titrated suspensions on first instar larvae. When tested at 3 X 10(5) conidia/cm2, only 2 strains, B. bassiana n. 297 and B. bassiana n. 326 killed 100% of larvae at 10 days post-exposure. In the same time their LD50 and their LD90 did not differ significantly. After 3 weeks, the mortality caused by either dose of spores of B. bassiana n. 297 was very high. In contrast, in the case of B. bassiana n. 326 mortality due to reduced doses remained at low rates. This laboratory study demonstrated that the isolate, B. bassiana n. 297 might have potential as microbial control agent against the assassin bugs.

[Influence of clay on microorganisms conservation. I. Ultrastructural study of the biodegradation of Beauveria bassiana (Bals). Vuill. (entomopathogenous hyphomycete) in soil (author's transl)]. / Effet de l'argile sur la conservation des microorganismes. I. Etude ultrastructurale de la biodégradation dans le sol de l'hypomycète entomopathogène Beauveria bassiana (Bals.) Vuill.

Blastospores of B. bassiana enclosed in fine mesh bags were submitted to soil microflora. Clay minerals coating fungal spores protected from by decreasing bacterial activity. The role of amoeba in biodegradation of fungi is also considered.